Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli.
The bacterial membrane serves both as a cell organelle and as a barrier for segregating the metabolically active cytoplasm from the extracellular milieu. Thus we can use plasmid vectors designed to produce a hybrid protein containing an efficient signal peptide coupled to the amino terminus of the cloned heterologous protein (secretion cloning vectors) for the production of proteins which are insoluble, proteolytically sensitive, or bacteriocidal when produced in the cytoplasm of Escherichia coli. We demonstrate that human granulocyte-macrophage colony stimulating factor can be isolated as an active species only after transport into the bacterial periplasm. Production of the protein in the bacterial cytoplasm is bacteriocidal. We also demonstrate that biologically active human interleukin 4 appears only after transport of the protein into the bacterial growth medium. The protein forms membrane-associated aggregates in the cytoplasm, and demonstrates an active but nonnative conformation when expressed in the periplasm. This may correlate with the affinity of the interleukin 4 molecule for negatively charged macromolecules, including bacterial membrane components and bacterial lipopolysaccharides, which may alter the folding pathway inside the cell.
Lundell
D
D
Biotechnology-Molecular Biology, Schering-Plough Research, Bloomfield, New Jersey 07003.
Lunn
C
C
Greenberg
R
R
Fossetta
J
J
Narula
S
S
Kastelein
R
R
Van Kimmenade
A
A
D000818:Animals; D000838:Anions / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D051379:Mice; D055503:Protein Multimerization / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013045:Species Specificity
D000838:Anions; D016207:Cytokines; D007376:Interleukin-2; D011994:Recombinant Proteins; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor